Tijekom stanične diobe, genetski materijal stanice se precizno dijeli u dvije stanice kćeri, cime mehanički i biološki upravlja mreža proteinskih filamenata koju nazivamo diobeno vreteno. Poznato je ...da ti proteinski filamenti, mikrotubuli, imaju elastična svojstva. Skupina mikrotubula, koju nazivamo kinetohorni mikrotubuli, djeluje silom tenzije na kromosome. Kinetohore su proteinski kompleksi koji su vezani na kromosome. Premda su sile koje djeluju u diobenom vretenu iscrpno istraživane, i dalje je nepoznato kako su one uravnotežene. Poznato je da se između sestrinskog para kinetohora nalazi još jedna skupina mikrotubula, koju mi ovdje nazivamo premosni mikrotubuli. Razvili smo model koji uključuje kinetohorne i premosne mikrotubule, te sile i momente sila koji djeluju me.u njima. Mikrotubuli su u modelu opisani kao tanki elastični štapovi koji se svijaju i uvijaju pod djelovanjem sila i momenata sila na njihovim krajevima. Iz oblika snopova mikrotubula, pomoću modela možemo odrediti kolike sile i momenti sila djeluju unutar diobenog vretena. Usporedbom sa izmjerenim oblicima predvidjeli smo da je sila na kinetohori Fk ~ 300 pN, a u polu F0 ~ 30 pN. Pokazali smo da premosni mikrotubuli omogućuju koegzistenciju sile kompresije u blizini polova te sile tenzije u blizini kinetohora, što je već niz godina otvoreno pitanje. Pokazali smo da je diobeno vreteno kiralno, te da su za kiralni oblik snopova mikrotubula odgovorni momenti sila u sustavu dva sestrinska kinetohorna vlakna i premosnog vlakna. Zaključujemo da uz sile u diobenom postoje i momenti sila, koje generiraju molekularni motori, te da oni zajedno sa silama određuju njegovu arhitekturu.
At the onset of division, the cell forms a network of protein filaments, mitotic spindle, which divides genetic material between the two nascent daughter cells. It is known that protein filaments, microtubules, behave as elastic objects. Forces that govern the division are exerted by k-fibres, bundles of microtubules which end at the kinetochores, protein complexes which mechanically couple microtubules to the chromosomes. Although forces that exist in mitotic spindle have been studied thoroughly, how they are balanced still remains elusive. It is known that nonkinetochore microtubules exist between the sister pairs of kinetochores, which we term bridging microtubules. We have developed a model that includes k-fibres and bridging fibre, as well as forces and torques acting between them. We have modelled the microtubule bundles as slender elastic rods that undergo bending and torsion due to the forces and torques exerted at its ends. By using our model and known shape of microtubule bundles, we can infer the forces and torques that are present in mitotic spindle. We have compared shapes obtained from theory and experiment, and predicted that the force at kinetochore is Fk ~ 300 pN and the force at pole is F0 ~ 30 pN. By using a theoretical model that includes the bridging fibre, we explained the coexistence of the tension at kinetochores and compression at poles, which has been an open question for a few years now. In addition, we show that mitotic spindle is chiral, and that torques acting on the bridging and k- fibres spindle are responsible for chiral shapes of microtubule bundles. We conclude that, in addition to forces, torques that are generated by the motor proteins exist in the spindle and determine its architecture.
Tijekom stanične diobe, genetski materijal stanice se precizno dijeli u dvije stanice kćeri, cime mehanički i biološki upravlja mreža proteinskih filamenata koju nazivamo diobeno vreteno. Poznato je da ti proteinski filamenti, mikrotubuli, imaju elastična svojstva. Skupina mikrotubula, koju nazivamo kinetohorni mikrotubuli, djeluje silom tenzije na kromosome. Kinetohore su proteinski kompleksi koji su vezani na kromosome. Premda su sile koje djeluju u diobenom vretenu iscrpno istraživane, i dalje je nepoznato kako su one uravnotežene. Poznato je da se između sestrinskog para kinetohora nalazi još jedna skupina mikrotubula, koju mi ovdje nazivamo premosni mikrotubuli. Razvili smo model koji uključuje kinetohorne i premosne mikrotubule, te sile i momente sila koji djeluju me.u njima. Mikrotubuli su u modelu opisani kao tanki elastični štapovi koji se svijaju i uvijaju pod djelovanjem sila i momenata sila na njihovim krajevima. Iz oblika snopova mikrotubula, pomoću modela možemo odrediti kolike sile i momenti sila djeluju unutar diobenog vretena. Usporedbom sa izmjerenim oblicima predvidjeli smo da je sila na kinetohori Fk ~ 300 pN, a u polu F0 ~ 30 pN. Pokazali smo da premosni mikrotubuli omogućuju koegzistenciju sile kompresije u blizini polova te sile tenzije u blizini kinetohora, što je već niz godina otvoreno pitanje. Pokazali smo da je diobeno vreteno kiralno, te da su za kiralni oblik snopova mikrotubula odgovorni momenti sila u sustavu dva sestrinska kinetohorna vlakna i premosnog vlakna. Zaključujemo da uz sile u diobenom postoje i momenti sila, koje generiraju molekularni motori, te da oni zajedno sa silama određuju njegovu arhitekturu.
In the epidermal cells of onion (Allium cepa L.) bulb scales the endoplasmic reticulum (ER) can be subdivided into three domains: a peripheral tubular network, cisternae, and long tubular strands. ...The latter are the form in which the ER is moved in onion cells. During cold treatment the arrangement of the three domains changes drastically. The cisternae and long tubular strands disintegrate into short ER tubules which show rapid agitational motion. Long-distance movement is inhibited. The peripheral tubular ER network is presumably retained during cold treatment. Rewarming of previously chilled bulb scales initiates the reorganization of the ER into the three domains. The ER is partly relocated during recovery from cold treatment. Redistribution and reorganization of the ER is not affected by the microtubule-destabilizing herbicides oryzalin and trifluralin (5 μM). Cytochalasin D (2 μM), however, inhibits not only the relocation of ER material, as is evident by the absence of long tubular ER strands, but also the movement of other cell organelles. The latter cluster on top of the cisternae in a manner which is characteristic of treatment with the actin-filament inhibitor. The array of actin filaments is similar in unstressed, cold-treated cells, and cells which recover from low temperatures in the presence of oryzalin or tap water alone. In the presence of cytochalasin D the actin filaments are severely fragmented. The results indicate that low temperatures most likely influence either the interaction of the force-generating system, probably myosin, with actin filaments, or the force-generating mechanism of the actomyosin-driven intracellular movement, but do not affect actin-filament integrity.
A phragmosome (PS) is a transvacuolar aggregation of cytoplasm that develops in the plane of future cytokinesis and is found specifically in highly vacuolated cells. Although protonemal cells of ...Adiantum capillus-veneris L. usually do not form a PS, a PS-like structure developed at the site of a preprophase band (PPB) of microtubules (MTs) when the nucleus and endoplasm were displaced from the division site by centrifugation, leaving a PPB in the cortical cytoplasm. The PS-like structure contained endoplasmic MTs, Factin, oil droplets and mitochondria. The structure did not develop when the cells were centrifuged before the formation of a PPB. Application of amiprophos-methyl (APM) before development of the PPB strongly inhibited the formation of the PS-like structure after centrifugation. The PS-like structure was dispersed after cytokinesis which occurred in the region of the displaced nucleus. Treatment with APM after the formation of the PS-like structure arrested the cell cycle at the M phase and inhibited the degradation of this structure. These results suggest that development of a PS-like structure is associated both with the formation of a PPB and with the stage of the cell cycle.
Arrangements of microfibrils (MFs) and microtubules (MTs) were examined in tracheary elements (TEs) of Pisum sativum L. and Commelina communis L. by production of replicas of cryo-sections, and by ...immunofluorescence microscopy, respectively. The secondary wall thickenings of TEs of Pisum and Commelina roots have pitted and latticed patterns, respectively. Most MFs in the pitted thickening of Pisum TEs retain a parallel alignment as they pass around the periphery of pits. However, some groups of MFs grow into the pits but then terminate at the edge of the thickening, indicating that cellulose-synthase complexes are inactivated in the plasma membrane under the pit. Microtubules of TEs of both Pisum and Commelina are localized under the secondary thickening and few MTs are detected in the areas between wall thickenings. In the presence of the MT-disrupting agent, amiprophosmethyl, cellulose and hemicellulose, which is specific to secondary thickening, are deposited in deformed patterns in TEs of Pisum roots, Pisum epicotyls and Commelina roots. This indicates that the localized deposition of hemicellulose as well as cellulose involves MTs. The deformed, but heterogeneous pattern of secondary thickening is still visible, indicating that MTs are involved in determining and maintaining the regular patterns of the secondary thickening but not the spatial heterogeneous pattern of the wall deposition. A working hypothesis for the formation of the secondary thickening is proposed.
Pulsed UV-A lasers implemented on regular light microscope systems are excellent for intracellular nanosurgery. Applying a careful optical implementation, the quality of nanosurgery based on ...plasma-induced ablation is dramatically increased over commercially available systems. We demonstrate the efficiency of a system by showing in vivo cytoskeleton nanosurgery and compare the performance to recent applications in intracellular surgery; in particular to femtosecond laser-based systems. We estimate that subnanosecond UV-A laser-based systems deposit at least an order of magnitude less energy than a typical femtosecond laser-based system to achieve a similar biological effect.
Intra- bzw. subzelluläre Nanochirurgie läßt sich hervorragend mit gepulsten UV-A Lasern implementieren. Eine sorgfältige Implementation der Einkopplung bewahrt das Diffraktionslimit und reduziert die eingespeiste Energie im Vergleich zu kommerziell verfügbaren Systemen dramatisch. Wir demonstrieren unsere Implementation beim Schneiden von Elementen des Zytoskeletts und vergleichen sie insbesondere mit Systemen, die Femtosekundenpulse verwenden. Nach unserer Schätzung liegt der Energieeintrag in die Probe mindestens eine Größenordnung unter der eines typischen Femtosekundenlaser-basierten Systems, um einen vergleichbaren biologischen Effekt zu erzielen.
We have studied the timing of preprophase band (PPB) development in the division cycle of onion (Allium cepa L.) root-tip cells by combinations of immunofluorescence microscopy of microtubules, ...microspectrophotometry of nuclear DNA, and autoradiography of 3Hthymidine incorporation during pulse-chase experiments. In normally grown onion root tips, every cell with a PPB had the G2 level of nuclear DNA. Some were in interphase, prior to chromatin condensation, and some had varying degrees of chromatin condensation, up to the stage of prophase at which the PPB-prophase spindle transition occurs. In addition, autoradiography showed that PPBs can be formed in cells which have just finished their S phase, and microspectrophotometry enabled us to detect a population of cells in G2 which had no PPBs, these presumably including cells which had left the division cycle. The effects of inhibitors of DNA synthesis showed that the formation of PPBs is not fully coupled to events of the nuclear cycle. Although the mitotic index decreased 6—10-fold to less than 0.5% when roots were kept in 20 μg·ml-1 aphidicolin for more than 8 h, the percentage of cells containing PPBs did not decrease in proportion: the number of cells in interphase with PPBs increased while the number in prophase decreased. Almost the same phenomena were observed in the presence of 100 μg·ml-1 5-aminouracil and 40 μg·ml-1 hydroxyurea. In controls, all cells with PPBs were in G2 or prophase, but in the presence of aphidicolin, 5-aminouracil or hydroxyurea, some of the interphase cells with PPBs were in the S phase or even in the G1 phase. We conclude that PPB formation normally occurs in G2 (in at least some cases very early in G2) and that this timing can be experimentally uncoupled from the timing of DNA duplication in the cell-division cycle. The result accords with other evidence indicating that the cytoplasmic events of cytokinesis are controlled in parallel to the nuclear cycle, rather than in an obligatorily coupled sequence.
